CLA is a generic term for various isomers of conjugated linoleic acid, of which only two isomers (the cis-9,trans-11 isomer, i.e. bovinic acid, and a trans-10,cis-12 isomer) are found to be biologically active. Synthetically produced, commercial CLA preparations generally contain all different isomers of CLA and only 40% of the c9,t11 isomer, whereas milk contains 80% of the c9,t11-18:2 isomer.
Several studies have reported that animal fats contain a fatty acid that, among other things, inhibits cancer in test animals, affects growth factors and may control the amount of fatty tissue in an organism. While studying hamburger beefs, Michael Pariza found that they contained a fatty acid that was assayed to be conjugated linoleic acid (CLA). It was found in animal tests that in the group of those fed with CLA-containing diet the number of mammary, stomach and colon cancer incidences was reduced as compared with the reference group (Pariza, M. W., Loretz, L. J., Storkson, J. M. and Holland, N. C., Mutagens and modulator of mutagenesis in fried ground beef, Cancer Res. (Suppl.), 1983, 43:2444s-2446s and Pariza, M. W. and Hargraves, W. A., A beef-derived mutagenesis modulator inhibits initiation of mouse epidermal tumors by 7,12-dimethylbenzanthracene, Carcinogenesis, 1985, 6:591-593). Also in tissue cultures of human cells, CLA has been capable of inhibiting development of cancer cells. The mechanism how it acts is still unknown, but CLA has been found to have an effect on various stages of tumorigenesis, many growth factors and also possibly on the metabolism of carcinogenic substances in the liver. It has also been suggested that CLA would act as an antioxidant (Ip, C., Chin, S. F., Scimeca, J. A. and Pariza, M. W., Mammary cancer prevention by conjugated dienoic derivatives of linoleic acid, Cancer Res., 1991, 51:6118-124), whereby the compound would protect cell membranes from the adverse effect of free radicals. The cholesterol-lowering effect of the compound has also been studied and found that the compound does not reduce the amount of the good high density lipoprotein (HDL) as the cholesterol-lowering drugs do (Lee, K. N., Kritchevsky, D. And Pariza, M. W., Conjugated linoleic acid and atherosclerosis in rabbits, Atherosclerosis, 1994, 108:19-25). CLA may also be beneficial to slimmers, because the compound has been found to split fatty tissues (Park et al., Changes in Body Composition in Mice during Feeding and Withdrawal of Conjugated Linoleic Acid, Lipids, 1999, 34:243-248).
Unconjugated linoleic acid, in turn, has been reported to have adverse effects, e.g. a stimulating effect on breast cancer. The antimicrobial effect of unconjugated linoleic acid is also commonly known.
CLA can be produced chemically or by enzymatic isomerization. Natural CLA is formed e.g. in the rumen of ruminant animals from polyunsaturated fatty acids as a result of biological activity of the bacterium Butyrivibrio fibrisolvens and is secreted therefrom both into milk and meat, which are found to be the best sources of CLA.
It has been found that the amount of CLA received from food has considerably decreased during the past decade. It is calculated in dietary analyses that in the 1970s an average diet contained about 0.45 g of CLA daily. As the use of milk and milk products has decreased, an average daily intake is currently 0.25 g of CLA. To increase natural CLA in food is highly essential in view of public health, because to double the CLA intake would reduce the risk of cancer, for instance, according to researches.
Several studies have reported on the importance of milk, in particular, of all foods as a source of CLA. For instance, according to a Finnish epidemiological survey (Knekt et al., oral statement) use of milk reduced the risk of breast cancer. Currently, CLA concentration in milk fat varies seasonally considerably (2.4 to 28.1 mg/g fat) depending on the quality of feeding.
Useful microbes in Intestinal tracts have been found to form CLA. Particularly, Butyrivibrio fibrisolvens bacterium occurring in the rumen and an isomerase enzyme thereof have been studied. However, this bacterium is so anaerobic that it is not possible to produce CLA therewith on an industrial scale, because the fully anaerobic conditions required by the strain are difficult and uneconomical to achieve (U.S. Pat. No. 5,856,149, Pariza et al.)
Propionibacterium acnes has also been found to produce CLA, but this pathogenic strain also produces a reductase enzyme which reduces the produced CLA further to other fatty acids (Verhulst et al., System. Appl. Microbiol., 9:12-15 (1987).
It is also commonly known that some propionic acid bacteria are capable of converting linoleic acid to the conjugated cis-9,trans11 form thereof.
Moreover, it is commonly known that the conversion of free linoleic acid to CLA is more efficient than that of the fatty acid in the form of a triglyceride. However, the free linoleic acid has an inhibitory effect on the growth of propionic acid bacteria even in relatively low concentrations, which has so far prevented the production of the conjugated linoleic acid, and particularly the cis-9,trans-11 form thereof, on a large scale.
U.S. Pat. No. 5,856,149 (Pariza and Yang) discloses a method of producing a cis-9,trans-11 fatty acid by conversion of unconjugated unsaturated (double bonds at positions 9 and 12) fatty acid with a strain of Lactobacillus reuterii, preferably L. reuterii PYR8. The publication describes isolation of CLA-producing strains, and states that only four of the 45 isolated strains had the desired linoleate isomerase activity and they were thus able to produce CLA from the linoleic acid. The researchers observed that the amount of CLA produced was in direct proportion to the amount of cells, and assume that the linoleate isomerase is an accumulating enzyme that has no functional significance in cell growth. The publication does not state an inhibitory effect of the linoleic acid on bacterial growth, and consequently, no solution is set forth to solve this problem.
In the article Production of conjugated linoleic add by dairy starter cultures, J. Appl. Microbiol, 85 (1998), pp. 95-102, J. Jiang, L. Björck and R. Fonden set forth that propionic acid bacteria are able to convert linoleic acid to CLA. Jiang et al. studied the ability of 19 different starter bacteria to convert the linoleic acid added to a growth medium to CLA after having observed that ripened cheeses contained higher levels of CLA than other milk products. They studied the ability of 7 lactobacillus strains, 4 lactococcus strains, 2 streptococcus strains and 6 propionic acid bacteria to produce CLA from linoleic acid in MRS, milk and Na-lactate media. In addition, different linoleic acid concentrations were studied by adding linoleic acid to MRS broth in an aqueous solution of Tween 80 detergent. Only a few propionic acid bacteria of the studied bacteria were observed to have bioconversion activity, three out of six strains, i.e. Propionibacterium freudenreichii ssp. freudenreichii PFF and PFF6 and P. freudenreichii ssp. shermanii PFS exhibited activity. The highest yield 265 μg/ml of CLA from the original linoleic acid concentration of 750 μg/ml was achieved with the PFF6 strain. Of the produced CLA, 70 to 90% was biologically active c9,t11 isomer. None of the lactobacilli, lactococci or streptococci was found to produce CLA.
Thus, the best propionic acid bacterium, the PFF6 strain, was capable of converting only 35% of the added linoleic acid to CLA with the technique described by Jiang et al. The researchers observed that the CLA production of propionic acid bacteria correlates positively with their tolerance to free linoleic acid. Thus, this study also confirmed the commonly known fact that the linoleic acid has an antimicrobial effect which inhibits the growth of bacteria. The publication states that the effect of antimicrobial fatty acids can be reduced by using surfactants, such as a detergent, e.g. Tween 80, or proteins. Studies on these lines have not been conducted, however, and the publication does not describe possible, useful techniques.
WO 99/29886, by J. Jiang, L. Björck and R. Fonden, is partly based on the research results set forth in the above-mentioned article. The application relates to the use of specific bacteria, useful in foodstuff applications, to produce CLA in vitro. In addition to Propionibacterium freudenreichii ssp. freudenreichii and P. freudenreichii ssp. shermanii, suitable bacteria also include Bifidobacterium breve. According to the publication, fermentation can be performed in the presence of an emulsifier, as examples are given Tween 80 and lecithin. However, the use of an emulsifier is not described in the examples of this publication, either, and the best result obtained is reported to be the same as in the above publication: 246.4 μg/ml of biologically active c9,t11 isomer was obtained from the original linoleic acid concentration of 750 μg/ml by using the strain PFF6. The yield is thus less than 33%.
Consequently, there is still an obvious need to provide new methods for producing conjugated linoleic acid with improved yields. When there is a desire to utilize the largest possible amounts of free linoleic acid as the starting material of CLA, the essential thing is how the problems associated with the antimicrobial effect of the free linoleic acid can be minimized or avoided.